Fc receptors (FcRs) are membrane receptors expressed on a number of immune effector cells. Upon interaction with target immunoglobulins, FcRs mediate a number of cellular responses, including, activation of cell mediated killing, induction of mediator release from the cell, uptake and destruction of antibody coated particles, and transport of immunoglobulins. Deo et al., 1997, Immunology Today 18:127-135. Further, it has been shown that antigen-presenting cells, e.g., macrophages and dendritic cells, undergo FcR mediated internalization of antigen-antibody complexes, allowing for antigen presentation and the consequent amplification of the immune response. As such, FcRs play a central role in development of antibody specificity and effector cell function. Deo et al., 1997, Immunology Today 18:127-135.
FcRs are defined by their specificity for immunoglobulin isotypes; Fc receptors for IgG antibodies are referred to as FcγR, for IgE as FcεR, for IgA as FcαR and so on. FcRn is a special class of Fc receptor found on neonatal cells and is responsible for, among other things, transporting maternal IgG from milk across the infants intestinal epithelial cells. Three subclasses of human gamma receptors have been identified: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Because each human FcγR subclass is encoded by two or three genes, and alternative RNA spicing leads to multiple transcripts, a broad diversity in Fcγ isoforms exists. The three genes encoding the human FcγRI subclass (FcγRIA, FcγRIB and FcγRIC) are clustered in region 1q21.1 of the long arm of chromosome 1; the genes encoding FcγRII isoforms (FcγRIIA, FcγRIIB and FcγRIIC) and the two genes encoding FcγRIII (FcγRIIIA and FcγRIIIB) are all clustered in region 1q22. FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J Lab. Clin. Med. 126:330-41 (1995).
Human FcγRI is a heteroligomeric complex composed of an α-chain and γ-chain. The α-chain is a 70-72 kDa glycoprotein having 3 extracellular C-2 Ig like domains, a 21 amino acid membrane domain and a charged cytoplasmic tail of 61 amino acids. van de Winkel et al., 1993, Immunology Today 14:215-221. The γ-chain is a homodimer that is involved in cell surface assembly and cell signaling into the interior of the cell. Each chain of γ homodimer includes a motif involved in cellular activation designated the ITAM motif. Human FcγRI binds monomeric IgG with high affinity (10−7-10−9M) through the action of the third extracellular C-2 domain.
FcγRII is a 40 kDa glycoprotein having two C2 set Ig-like extracellular domains, a 27-29 amino acid transmembrane domain, and a cytoplasmic domain having variable length, from 44 to 76 amino acids. There are six known isoforms of the human FcγRII, differing for the most part in their heterogeneous cytoplasmic domains. Human FcγRIIA includes an ITAM motif in the cytoplasmic region of the molecule, and upon crosslinking of the receptor this motif is associated with cellular activation. In contrast, human FcγRIIB includes an inhibitory motif in its cytoplasmic region designated ITIM. When the FcγRIIB is crosslinked, cellular activation is inhibited. In general, FcγRII binds monomeric IgG poorly (>107 M−1), but has high affinity for complexed IgG.
Human FcγRIII has two major isoforms, FcγRIIIA and FcγRIIIB, both isoforms are between 50 to 80 kDa, having two C2 Ig-like extracellular domains. The FcγRIIIA α-chain is anchored to the membrane by a 25 amino acid transmembrane domain, while FcγRIIIB is linked to the membrane via a glycosyl phosphatidyl-inositol (GPI) anchor. Human FcγRIIIA is a heteroligomeric complex with the α-chain complexed with a heterodimeric γ-δ (gamma-delta) chain or γ-γ chain. The γ-chain includes a cytoplasmic tail with an ITAM motif. The γ-chain is homologous to the α-chain and is also involved in cell signaling and cell surface assembly. The γ-δ (gamma-delta) chain also includes an ITAM motif in its cytoplasmic region. In both cases, the FcγRIII binds monomeric IgG with low affinity, and binds complexed IgG with high affinity.
Human FcRn is a heterodimer composed of a β-2 microglobulin chain and a α chain. The β-2 microglobulin chain is approximately 15 kDa and is similar to the β-2 microglobulin chain present in MHC class I heterodimers. The presence of a β-2 microglobulin chain in FcRn makes it the only known Fc receptor to fall within the MHC class I family of proteins. Ghetie et al., 1997 Immunology Today 18(12):592-598. The a chain is a 37-40 kDa integral membrane glycoprotein having a single glycosylation site. Evidence suggests that FcRn is involved in transferring maternal IgG across the neonatal gut and in regulating serum IgG levels. FcRn is also found in adults on many tissues.
As discussed above, human FcγRs, with the exception of FcγRIIB, contain a cytoplasmic ˜26 amino acid immunoreceptor tyrosine-based activation motif (ITAM). It is believed that this motif is involved in cell signaling and effector cell function. Crosslinking of FcγRs may lead to the phosphorylation of tyrosine residues within the ITAM motif by src-family tyrosine kinases (PTKs), followed by association and activation of the phosphorylated ITAM motif with syk-family PTKs. Deo et al., 1997, Immunology Today 18:127-135. Once activated, a poorly understood signaling cascade is translated into biological responses.
Human FcγRIIB members contain a distinct 13 amino acid immuno-receptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domain. Human FcγRIIB is expressed on B lymphocytes and binds to IgG complexes. However, rather than activating cells, crosslinking of the IIB receptor results in a signal inhibiting B cell activation and antibody secretion. (Camigorea et al., 1992, Cytoplasmic Domain Heterogeneity and Function of IgG Receptors in B Lymphocytes, Science 256:1808.)
Because of the central role of FcγR as a trigger molecule in numerous immune responses, it has become a target for developing potential therapeutics. For example, several ongoing clinical trials are based on activating a cancer patient's effector cells by treating the patient with tumor-specific monoclonal antibodies (Mabs). These studies have shown that the tumor-specific antibodies mediate their effects in part through FcγR binding, and subsequent effector cell activity. Adams et al., 1984, Proc. Natl. Acad. Sci. 81:3506-3510; Takahashi et al., 1995, Gastroenterology 108:172-182; Riethmeuller et al., 1994, Lancet 343:1177-1183, Clynes, R. A., Towers, T. L., Presta, L. G., and Ravetch, J. V., 2000, Nature Med. 6:443-446. Further, a novel series of bispecific molecule antibodies (BSMs), molecules engineered to have one arm specific for a tumor cell and the other arm specific for a target FcγR, are in clinical trials to specifically target a tumor for FcγR mediated, effector cell destruction of the tumor cells. Valone et al., 1995, J. Clin. Oncol. 13:2281-2292; Repp et al., 1995, Hematother 4:415-421. In addition, FcγRs can be used as therapeutic targets in a number of infectious diseases, and for that matter, a number of autoimmune disorders. With regard to infectious diseases, BSMs are being developed to target any number of microorganisms to a patient's FcγR expressing effector cells (Deo et al., 1997, Immunology Today 18:127-135), while soluble FcγRs have been used to inhibit the Arthus reaction, and FcγR blocking agents have been used to reduce the severity of several autoimmune disorders. Ierino et al., 1993, J. Exp. Med. 178:1617-1628; Debre et al., 1993, Lancet 342:945-949.
As antibodies have become increasingly used as therapeutic agents, there is a need to develop animal models for evaluating the toxicity, efficacy and pharmacokinetics of such therapeutic agents. In addition to rodent models for evaluating efficacy of antibody therapeutics, primate models have been used for evaluation of therapeutic antibody pharmacokinetics, toxicity, and efficacy (Anderson, D. R., Grillo-Lopez, A., Varns, C., Chambers, K. S., and Hanna, N. (1997) Biochem. Soc. Trans. 25, 705-708). However, there is only sparse information available regarding the interaction of human antibodies with primate Fcy receptors and the effects of this interaction on interpretation of pharmacokinetic, toxicity, and efficacy studies in primates.
Although many advances have been made in elucidating FcγR activity and identifying and engineering FcγR ligands, there still remains a need in the art to identify other FcγRs and to identify and engineer other FcγR ligands, both activating and inhibiting. These new receptors and receptor ligands possess potential therapeutic value in a number of disease states, including, the destruction of tumor cells and infectious material, as well as in blocking portions of the immune response involved in several autoimmune disorders. As antibodies and other FcγR ligands are used as therapeutic agents, there is also a need to develop models to test the efficacy, toxicity, and pharmacokinetics of these therapeutic agents, especially in vivo.